data_50208 ####################### # Entry information # ####################### save_entry_information _Saveframe_category entry_information _Entry_title ; Purification of native CCL7 and its functional interaction with selected chemokine receptors ; _BMRB_accession_number 50208 _BMRB_flat_file_name bmr50208.str _Entry_type original _Submission_date 2020-03-07 _Accession_date 2020-03-07 _Entry_origination author _NMR_STAR_version 2.1.1 _Experimental_method NMR _Details ; Chemokine receptors form a major sub-family of G protein-coupled receptors (GPCRs) and they are involved in a number of cellular and physiological processes related to our immune response and regulation. A better structural understanding of ligand-binding, activation, signaling and regulation of chemokine receptors is very important to design potentially therapeutic interventions for human disorders arising from aberrant chemokine signaling. One of the key limitations in probing the structural details of chemokine receptors is the availability of large amounts of purified, homogenous and fully functional chemokine ligands, and the commercially available products, are not affordable for in-depth structural studies. Moreover, production of uniformly isotope-labeled chemokines, for example, suitable for NMR-based structural investigation, also remains challenging. Here, we have designed a streamlined approach to express and purify the human chemokine CCL7 as well as its 15N-, 15N/13C-, 2H/15N/13C- isotope-labeled derivatives, at milligram levels using E. coli expression system. Purified CCL7 not only maintains a well-folded three-dimensional structure as analyzed using circular dichroism and 1H/15N NMR but it also induces coupling of heterotrimeric G-proteins and b-arrestins for selected chemokine receptors in cellular system. We compared cAMP response induced by histidine tagged CCL7 and native CCL7 and found that modification of the N-terminus of CCL7 compromises its functionality. Our strategy presented here may be applicable to other chemokines and therefore, provide a potentially generic and cost-effective approach to produce chemokines in large amounts for functional and structural studies. ; loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Goncharuk Marina V. . 2 Roy Debarati . . 3 Dubinnyi Maxim A. . 4 Nadezhdin Kirill D. . 5 Srivastava Ashish . . 6 Baidya Mithu . . 7 Dwivedi-Agnihotri Hemlata . . 8 Arseniev Alexander S. . 9 Shukla Arun K. . stop_ loop_ _Saveframe_category_type _Saveframe_category_type_count assigned_chemical_shifts 1 stop_ loop_ _Data_type _Data_type_count "1H chemical shifts" 464 "15N chemical shifts" 84 stop_ loop_ _Revision_date _Revision_keyword _Revision_author _Revision_detail 2020-08-25 original BMRB . stop_ loop_ _Related_BMRB_accession_number _Relationship 4177 'This entry was used to transfer NMR assignment' 50207 'Native human chemokine CCL7' stop_ _Original_release_date 2020-03-20 save_ ############################# # Citation for this entry # ############################# save_citations_1 _Saveframe_category entry_citation _Citation_full . _Citation_title ; Purification of native CCL7 and its functional interaction with selected chemokine receptors ; _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 32145391 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Goncharuk Marina V. . 2 Roy Debarati . . 3 Dubinnyi Maxim A. . 4 Nadezhdin Kirill D. . 5 Srivastava Ashish . . 6 Baidya Mithu . . 7 Dwivedi-Agnihotri Hemlata . . 8 Arseniev Alexander S. . 9 Shukla Arun K. . stop_ _Journal_abbreviation 'Protein Expr. Purif.' _Journal_name_full 'Protein Expression and Purification' _Journal_volume . _Journal_issue . _Journal_CSD . _Book_chapter_title . _Book_volume . _Book_series . _Book_ISBN . _Conference_state_province . _Conference_abstract_number . _Page_first . _Page_last . _Year 2020 _Details . loop_ _Keyword Arrestins 'Atypical chemokine receptors' 'Biased agonism' Chemokine G-protein GPCR 'Isotope labeling; NMR spectroscopy' 'Recombinant protein' stop_ save_ ################################## # Molecular system description # ################################## save_assembly_1 _Saveframe_category molecular_system _Mol_system_name 'His6-tagged human chemokine CCL7' _Enzyme_commission_number . loop_ _Mol_system_component_name _Mol_label HisCCL7 $entity_1 stop_ _System_molecular_weight 10054 _System_physical_state native _System_oligomer_state ? _System_paramagnetic no _System_thiol_state . _Database_query_date . _Details 'His6-tagged human chemokine CCL7' save_ ######################## # Monomeric polymers # ######################## save_entity_1 _Saveframe_category monomeric_polymer _Mol_type polymer _Mol_polymer_class protein _Name_common entity_1 _Molecular_mass . _Mol_thiol_state 'all disulfide bound' loop_ _Biological_function 'Chemotactic factor that attracts monocytes' stop_ _Details . ############################## # Polymer residue sequence # ############################## _Residue_count 85 _Mol_residue_sequence ; MHHHHHHGSQPVGINTSTTC CYRFINKKIPKQRLESYRRT TSSHCPREAVIFKTKLDKEI CADPTQKWVQDFMKHLDKKT QTPKL ; loop_ _Residue_seq_code _Residue_author_seq_code _Residue_label 1 -8 MET 2 -7 HIS 3 -6 HIS 4 -5 HIS 5 -4 HIS 6 -3 HIS 7 -2 HIS 8 -1 GLY 9 0 SER 10 1 GLN 11 2 PRO 12 3 VAL 13 4 GLY 14 5 ILE 15 6 ASN 16 7 THR 17 8 SER 18 9 THR 19 10 THR 20 11 CYS 21 12 CYS 22 13 TYR 23 14 ARG 24 15 PHE 25 16 ILE 26 17 ASN 27 18 LYS 28 19 LYS 29 20 ILE 30 21 PRO 31 22 LYS 32 23 GLN 33 24 ARG 34 25 LEU 35 26 GLU 36 27 SER 37 28 TYR 38 29 ARG 39 30 ARG 40 31 THR 41 32 THR 42 33 SER 43 34 SER 44 35 HIS 45 36 CYS 46 37 PRO 47 38 ARG 48 39 GLU 49 40 ALA 50 41 VAL 51 42 ILE 52 43 PHE 53 44 LYS 54 45 THR 55 46 LYS 56 47 LEU 57 48 ASP 58 49 LYS 59 50 GLU 60 51 ILE 61 52 CYS 62 53 ALA 63 54 ASP 64 55 PRO 65 56 THR 66 57 GLN 67 58 LYS 68 59 TRP 69 60 VAL 70 61 GLN 71 62 ASP 72 63 PHE 73 64 MET 74 65 LYS 75 66 HIS 76 67 LEU 77 68 ASP 78 69 LYS 79 70 LYS 80 71 THR 81 72 GLN 82 73 THR 83 74 PRO 84 75 LYS 85 76 LEU stop_ _Sequence_homology_query_date . _Sequence_homology_query_revised_last_date . loop_ _Database_name _Database_accession_code _Database_entry_mol_name _Sequence_query_to_submitted_percentage _Sequence_subject_length _Sequence_identity _Sequence_positive _Sequence_homology_expectation_value SP P80098 . . . . . . stop_ save_ #################### # Natural source # #################### save_natural_source_1 _Saveframe_category natural_source loop_ _Mol_label _Organism_name_common _NCBI_taxonomy_ID _Superkingdom _Kingdom _Genus _Species $entity_1 Human 9606 Eukaryota Metazoa Homo sapiens stop_ save_ ######################### # Experimental source # ######################### save_experimental_source_1 _Saveframe_category experimental_source loop_ _Mol_label _Production_method _Host_organism_name_common _Genus _Species _Strain _Vector_type _Vector_name $entity_1 'recombinant technology' . Escherichia coli BL21(DE3)pLysS plasmid pGEMEX-1(his6) stop_ save_ ##################################### # Sample contents and methodology # ##################################### ######################## # Sample description # ######################## save_sample_1 _Saveframe_category sample _Sample_type solution _Details ; 303 oK pH 5.1 (KPi 6.12 titrated by d4-AcOD) 20mM KPi 0.4mM MES 7 mM NaCl ; loop_ _Mol_label _Concentration_value _Concentration_value_units _Isotopic_labeling $entity_1 0.8 mM '[U-100% 15N]' KPi 20 mM 'natural abundance' MES 0.4 mM 'natural abundance' NaCl 7 mM 'natural abundance' stop_ save_ ############################ # Computer software used # ############################ save_software_1 _Saveframe_category software _Name TOPSPIN _Version 3.0 loop_ _Task collection stop_ _Details . save_ save_software_2 _Saveframe_category software _Name CARA _Version 1.9.1.7 loop_ _Task 'chemical shift assignment' stop_ _Details . save_ ######################### # Experimental detail # ######################### ################################## # NMR Spectrometer definitions # ################################## save_NMR_spectrometer_1 _Saveframe_category NMR_spectrometer _Manufacturer Bruker _Model Avance _Field_strength 700 _Details 'Warm probe 5 mm PATXI' save_ save_NMR_spectrometer_2 _Saveframe_category NMR_spectrometer _Manufacturer Bruker _Model 'Avance III' _Field_strength 800 _Details 'Cryoprobe 5 mm CPTCI' save_ ############################# # NMR applied experiments # ############################# save_1D_1H_1 _Saveframe_category NMR_applied_experiment _Experiment_name '1D 1H' _Sample_label $sample_1 save_ save_2D_1H-15N_HSQC_2 _Saveframe_category NMR_applied_experiment _Experiment_name '2D 1H-15N HSQC' _Sample_label $sample_1 save_ save_3D_1H-15N_TOCSY_3 _Saveframe_category NMR_applied_experiment _Experiment_name '3D 1H-15N TOCSY' _Sample_label $sample_1 save_ save_3D_1H-15N_NOESY_4 _Saveframe_category NMR_applied_experiment _Experiment_name '3D 1H-15N NOESY' _Sample_label $sample_1 save_ ####################### # Sample conditions # ####################### save_sample_conditions_1 _Saveframe_category sample_conditions _Details . loop_ _Variable_type _Variable_value _Variable_value_error _Variable_value_units 'ionic strength' 0.007 . M pH 5.1 . pH pressure 1 . atm temperature 303 . K stop_ save_ #################### # NMR parameters # #################### ############################## # Assigned chemical shifts # ############################## ################################ # Chemical shift referencing # ################################ save_chem_shift_reference_1 _Saveframe_category chemical_shift_reference _Details . loop_ _Mol_common_name _Atom_type _Atom_isotope_number _Atom_group _Chem_shift_units _Chem_shift_value _Reference_method _Reference_type _External_reference_sample_geometry _External_reference_location _External_reference_axis _Indirect_shift_ratio water H 1 protons ppm 4.7 internal direct . . . 1 water N 15 protons ppm 4.7 internal indirect . . . 1 stop_ save_ ################################### # Assigned chemical shift lists # ################################### ################################################################### # Chemical Shift Ambiguity Index Value Definitions # # # # The values other than 1 are used for those atoms with different # # chemical shifts that cannot be assigned to stereospecific atoms # # or to specific residues or chains. # # # # Index Value Definition # # # # 1 Unique (including isolated methyl protons, # # geminal atoms, and geminal methyl # # groups with identical chemical shifts) # # (e.g. ILE HD11, HD12, HD13 protons) # # 2 Ambiguity of geminal atoms or geminal methyl # # proton groups (e.g. ASP HB2 and HB3 # # protons, LEU CD1 and CD2 carbons, or # # LEU HD11, HD12, HD13 and HD21, HD22, # # HD23 methyl protons) # # 3 Aromatic atoms on opposite sides of # # symmetrical rings (e.g. TYR HE1 and HE2 # # protons) # # 4 Intraresidue ambiguities (e.g. LYS HG and # # HD protons or TRP HZ2 and HZ3 protons) # # 5 Interresidue ambiguities (LYS 12 vs. LYS 27) # # 6 Intermolecular ambiguities (e.g. ASP 31 CA # # in monomer 1 and ASP 31 CA in monomer 2 # # of an asymmetrical homodimer, duplex # # DNA assignments, or other assignments # # that may apply to atoms in one or more # # molecule in the molecular assembly) # # 9 Ambiguous, specific ambiguity not defined # # # ################################################################### save_assigned_chemical_shifts_1 _Saveframe_category assigned_chemical_shifts _Details . loop_ _Software_label $software_2 stop_ loop_ _Experiment_label '1D 1H' '2D 1H-15N HSQC' '3D 1H-15N TOCSY' '3D 1H-15N NOESY' stop_ loop_ _Sample_label $sample_1 stop_ _Sample_conditions_label $sample_conditions_1 _Chem_shift_reference_set_label $chem_shift_reference_1 _Mol_system_component_name HisCCL7 _Text_data_format . _Text_data . loop_ _Atom_shift_assign_ID _Residue_author_seq_code _Residue_seq_code _Residue_label _Atom_name _Atom_type _Chem_shift_value _Chem_shift_value_error _Chem_shift_ambiguity_code 1 -1 8 GLY H H 8.524 0.020 1 2 -1 8 GLY HA2 H 3.998 0.020 1 3 -1 8 GLY HA3 H 3.998 0.020 1 4 -1 8 GLY N N 110.735 0.3 1 5 0 9 SER H H 8.321 0.020 1 6 0 9 SER HA H 4.485 0.020 1 7 0 9 SER HB2 H 3.856 0.020 1 8 0 9 SER HB3 H 3.856 0.020 1 9 0 9 SER N N 115.737 0.3 1 10 1 10 GLN H H 8.369 0.020 1 11 1 10 GLN HA H 4.667 0.020 1 12 1 10 GLN HB2 H 1.942 0.020 2 13 1 10 GLN HB3 H 2.090 0.020 2 14 1 10 GLN HG2 H 2.394 0.020 1 15 1 10 GLN HG3 H 2.394 0.020 1 16 1 10 GLN HE21 H 7.497 0.020 1 17 1 10 GLN HE22 H 6.830 0.020 1 18 1 10 GLN N N 122.890 0.3 1 19 1 10 GLN NE2 N 112.186 0.3 1 20 3 12 VAL H H 8.281 0.020 1 21 3 12 VAL HA H 4.108 0.020 1 22 3 12 VAL HB H 2.046 0.020 1 23 3 12 VAL HG1 H 0.970 0.020 2 24 3 12 VAL HG2 H 0.950 0.020 2 25 3 12 VAL N N 120.404 0.3 1 26 4 13 GLY HA2 H 3.916 0.020 2 27 4 13 GLY HA3 H 4.014 0.020 2 28 4 13 GLY N N 112.427 0.3 1 29 5 14 ILE H H 7.959 0.020 1 30 5 14 ILE HA H 4.185 0.020 1 31 5 14 ILE HB H 1.837 0.020 1 32 5 14 ILE HG12 H 1.410 0.020 2 33 5 14 ILE HG13 H 1.160 0.020 2 34 5 14 ILE HG2 H 0.890 0.020 1 35 5 14 ILE HD1 H 0.830 0.020 1 36 5 14 ILE N N 119.730 0.3 1 37 6 15 ASN H H 8.518 0.020 1 38 6 15 ASN HA H 4.799 0.020 1 39 6 15 ASN HB2 H 2.890 0.020 2 40 6 15 ASN HB3 H 2.770 0.020 2 41 6 15 ASN HD21 H 7.561 0.020 1 42 6 15 ASN HD22 H 6.891 0.020 1 43 6 15 ASN N N 122.156 0.3 1 44 6 15 ASN ND2 N 112.815 0.3 1 45 7 16 THR H H 8.165 0.020 1 46 7 16 THR HA H 4.360 0.020 1 47 7 16 THR HB H 4.280 0.020 1 48 7 16 THR HG2 H 1.193 0.020 1 49 7 16 THR N N 114.359 0.3 1 50 8 17 SER H H 8.331 0.020 1 51 8 17 SER HA H 4.550 0.020 1 52 8 17 SER HB2 H 3.930 0.020 2 53 8 17 SER HB3 H 3.880 0.020 2 54 8 17 SER N N 117.637 0.3 1 55 9 18 THR H H 8.399 0.020 1 56 9 18 THR HA H 4.360 0.020 1 57 9 18 THR HB H 4.169 0.020 1 58 9 18 THR HG2 H 1.150 0.020 1 59 9 18 THR N N 116.966 0.3 1 60 10 19 THR H H 8.224 0.020 1 61 10 19 THR HA H 4.332 0.020 1 62 10 19 THR HB H 4.140 0.020 1 63 10 19 THR HG2 H 1.220 0.020 1 64 10 19 THR N N 118.298 0.3 1 65 11 20 CYS H H 8.127 0.020 1 66 11 20 CYS HA H 4.964 0.020 1 67 11 20 CYS HB2 H 2.764 0.020 2 68 11 20 CYS HB3 H 2.540 0.020 2 69 11 20 CYS N N 118.451 0.3 1 70 12 21 CYS H H 8.317 0.020 1 71 12 21 CYS HA H 4.610 0.020 1 72 12 21 CYS HB2 H 2.410 0.020 2 73 12 21 CYS HB3 H 2.720 0.020 2 74 12 21 CYS N N 118.581 0.3 1 75 14 23 ARG H H 7.407 0.020 1 76 14 23 ARG HA H 4.305 0.020 1 77 14 23 ARG HB2 H 1.767 0.020 2 78 14 23 ARG HB3 H 1.660 0.020 2 79 14 23 ARG HG2 H 1.500 0.020 2 80 14 23 ARG HG3 H 1.040 0.020 2 81 14 23 ARG HD2 H 3.184 0.020 1 82 14 23 ARG HD3 H 3.184 0.020 1 83 14 23 ARG HE H 7.180 0.020 1 84 14 23 ARG N N 118.893 0.3 1 85 15 24 PHE H H 8.530 0.020 1 86 15 24 PHE HA H 4.798 0.020 1 87 15 24 PHE HB2 H 2.900 0.020 2 88 15 24 PHE HB3 H 3.198 0.020 2 89 15 24 PHE HD1 H 7.030 0.020 1 90 15 24 PHE HD2 H 7.030 0.020 1 91 15 24 PHE HE1 H 7.200 0.020 1 92 15 24 PHE HE2 H 7.200 0.020 1 93 15 24 PHE HZ H 7.120 0.020 1 94 15 24 PHE N N 121.139 0.3 1 95 16 25 ILE H H 8.682 0.020 1 96 16 25 ILE HA H 4.410 0.020 1 97 16 25 ILE HB H 2.180 0.020 1 98 16 25 ILE HG12 H 1.620 0.020 2 99 16 25 ILE HG13 H 1.730 0.020 2 100 16 25 ILE HG2 H 1.250 0.020 1 101 16 25 ILE HD1 H 1.050 0.020 1 102 16 25 ILE N N 121.993 0.3 1 103 17 26 ASN H H 8.654 0.020 1 104 17 26 ASN HA H 5.060 0.020 1 105 17 26 ASN HB2 H 2.940 0.020 1 106 17 26 ASN HB3 H 2.940 0.020 1 107 17 26 ASN HD21 H 7.659 0.020 1 108 17 26 ASN HD22 H 6.960 0.020 1 109 17 26 ASN N N 123.127 0.3 1 110 17 26 ASN ND2 N 112.443 0.3 1 111 18 27 LYS H H 7.440 0.020 1 112 18 27 LYS HA H 4.280 0.020 1 113 18 27 LYS HB2 H 1.710 0.020 2 114 18 27 LYS HB3 H 1.590 0.020 2 115 18 27 LYS HG2 H 1.290 0.020 1 116 18 27 LYS HG3 H 1.290 0.020 1 117 18 27 LYS HD2 H 1.590 0.020 1 118 18 27 LYS HD3 H 1.590 0.020 1 119 18 27 LYS HE2 H 2.950 0.020 1 120 18 27 LYS HE3 H 2.950 0.020 1 121 18 27 LYS N N 119.769 0.3 1 122 19 28 LYS H H 7.696 0.020 1 123 19 28 LYS HA H 2.490 0.020 1 124 19 28 LYS HB2 H 1.050 0.020 2 125 19 28 LYS HB3 H 0.990 0.020 2 126 19 28 LYS HG2 H 0.700 0.020 2 127 19 28 LYS HG3 H 0.530 0.020 2 128 19 28 LYS HD2 H 1.350 0.020 1 129 19 28 LYS HD3 H 1.350 0.020 1 130 19 28 LYS HE2 H 2.760 0.020 1 131 19 28 LYS HE3 H 2.760 0.020 1 132 19 28 LYS N N 120.622 0.3 1 133 20 29 ILE H H 6.100 0.020 1 134 20 29 ILE HA H 4.320 0.020 1 135 20 29 ILE HB H 1.640 0.020 1 136 20 29 ILE HG12 H 1.470 0.020 2 137 20 29 ILE HG13 H 1.050 0.020 2 138 20 29 ILE HG2 H 0.840 0.020 1 139 20 29 ILE HD1 H 0.830 0.020 1 140 20 29 ILE N N 122.500 0.3 1 141 22 31 LYS H H 8.309 0.020 1 142 22 31 LYS HA H 3.320 0.020 1 143 22 31 LYS HB2 H 1.364 0.020 2 144 22 31 LYS HB3 H 1.222 0.020 2 145 22 31 LYS HG2 H 0.690 0.020 2 146 22 31 LYS HG3 H 0.880 0.020 2 147 22 31 LYS HD2 H 1.090 0.020 2 148 22 31 LYS HD3 H 1.150 0.020 2 149 22 31 LYS HE2 H 2.500 0.020 1 150 22 31 LYS HE3 H 2.500 0.020 1 151 22 31 LYS N N 124.457 0.3 1 152 23 32 GLN H H 8.613 0.020 1 153 23 32 GLN HA H 4.171 0.020 1 154 23 32 GLN HB2 H 2.036 0.020 1 155 23 32 GLN HB3 H 2.036 0.020 1 156 23 32 GLN HG2 H 2.430 0.020 2 157 23 32 GLN HG3 H 2.370 0.020 2 158 23 32 GLN HE21 H 7.560 0.020 1 159 23 32 GLN HE22 H 6.890 0.020 1 160 23 32 GLN N N 116.350 0.3 1 161 23 32 GLN NE2 N 111.200 0.3 1 162 24 33 ARG H H 7.798 0.020 1 163 24 33 ARG HA H 4.318 0.020 1 164 24 33 ARG HB2 H 2.043 0.020 2 165 24 33 ARG HB3 H 1.780 0.020 2 166 24 33 ARG HG2 H 1.660 0.020 1 167 24 33 ARG HG3 H 1.660 0.020 1 168 24 33 ARG HD2 H 3.130 0.020 2 169 24 33 ARG HD3 H 3.220 0.020 2 170 24 33 ARG HE H 7.189 0.020 1 171 24 33 ARG N N 116.260 0.3 1 172 24 33 ARG NE N 84.598 0.3 1 173 25 34 LEU H H 7.542 0.020 1 174 25 34 LEU HA H 4.318 0.020 1 175 25 34 LEU HB2 H 1.896 0.020 2 176 25 34 LEU HB3 H 1.260 0.020 2 177 25 34 LEU HG H 1.560 0.020 1 178 25 34 LEU HD1 H 0.251 0.020 2 179 25 34 LEU HD2 H 0.504 0.020 2 180 25 34 LEU N N 119.131 0.3 1 181 26 35 GLU H H 9.113 0.020 1 182 26 35 GLU HA H 4.621 0.020 1 183 26 35 GLU HB2 H 1.930 0.020 2 184 26 35 GLU HB3 H 1.730 0.020 2 185 26 35 GLU HG2 H 2.210 0.020 1 186 26 35 GLU HG3 H 2.210 0.020 1 187 26 35 GLU N N 120.295 0.3 1 188 27 36 SER H H 8.042 0.020 1 189 27 36 SER HA H 4.785 0.020 1 190 27 36 SER HB2 H 4.020 0.020 2 191 27 36 SER HB3 H 4.080 0.020 2 192 27 36 SER N N 109.150 0.3 1 193 28 37 TYR H H 8.608 0.020 1 194 28 37 TYR HA H 5.730 0.020 1 195 28 37 TYR HB2 H 2.420 0.020 2 196 28 37 TYR HB3 H 2.900 0.020 2 197 28 37 TYR HD1 H 6.770 0.020 1 198 28 37 TYR HD2 H 6.770 0.020 1 199 28 37 TYR HE1 H 6.860 0.020 1 200 28 37 TYR HE2 H 6.860 0.020 1 201 28 37 TYR N N 115.680 0.3 1 202 29 38 ARG H H 8.535 0.020 1 203 29 38 ARG HA H 4.580 0.020 1 204 29 38 ARG HB2 H 1.822 0.020 2 205 29 38 ARG HB3 H 1.670 0.020 2 206 29 38 ARG HG2 H 1.350 0.020 1 207 29 38 ARG HG3 H 1.350 0.020 1 208 29 38 ARG HD2 H 3.050 0.020 2 209 29 38 ARG HD3 H 3.170 0.020 2 210 29 38 ARG HE H 7.496 0.020 1 211 29 38 ARG N N 117.238 0.3 1 212 29 38 ARG NE N 85.375 0.3 1 213 30 39 ARG H H 8.849 0.020 1 214 30 39 ARG HA H 5.180 0.020 1 215 30 39 ARG HB2 H 1.730 0.020 2 216 30 39 ARG HB3 H 2.071 0.020 2 217 30 39 ARG HG2 H 1.800 0.020 2 218 30 39 ARG HG3 H 1.650 0.020 2 219 30 39 ARG HD2 H 3.270 0.020 1 220 30 39 ARG HD3 H 3.270 0.020 1 221 30 39 ARG HE H 7.406 0.020 1 222 30 39 ARG N N 121.032 0.3 1 223 30 39 ARG NE N 84.294 0.3 1 224 31 40 THR H H 8.398 0.020 1 225 31 40 THR HA H 4.600 0.020 1 226 31 40 THR HB H 4.590 0.020 1 227 31 40 THR HG2 H 1.240 0.020 1 228 31 40 THR N N 113.152 0.3 1 229 32 41 THR H H 8.303 0.020 1 230 32 41 THR HA H 4.510 0.020 1 231 32 41 THR HB H 4.309 0.020 1 232 32 41 THR HG2 H 1.210 0.020 1 233 32 41 THR N N 113.972 0.3 1 234 33 42 SER H H 8.417 0.020 1 235 33 42 SER HA H 4.500 0.020 1 236 33 42 SER HB2 H 4.020 0.020 2 237 33 42 SER HB3 H 3.910 0.020 2 238 33 42 SER N N 117.750 0.3 1 239 34 43 SER H H 8.505 0.020 1 240 34 43 SER HA H 4.320 0.020 1 241 34 43 SER HB2 H 3.860 0.020 1 242 34 43 SER HB3 H 3.860 0.020 1 243 34 43 SER N N 119.348 0.3 1 244 35 44 HIS H H 8.136 0.020 1 245 35 44 HIS HA H 4.690 0.020 1 246 35 44 HIS HB2 H 3.110 0.020 2 247 35 44 HIS HB3 H 3.350 0.020 2 248 35 44 HIS N N 116.783 0.3 1 249 36 45 CYS H H 7.674 0.020 1 250 36 45 CYS HA H 5.180 0.020 1 251 36 45 CYS HB2 H 2.680 0.020 2 252 36 45 CYS HB3 H 3.360 0.020 2 253 36 45 CYS N N 117.904 0.3 1 254 38 47 ARG H H 7.150 0.020 1 255 38 47 ARG HA H 4.541 0.020 1 256 38 47 ARG HB2 H 1.693 0.020 2 257 38 47 ARG HB3 H 1.882 0.020 2 258 38 47 ARG HG2 H 1.530 0.020 1 259 38 47 ARG HG3 H 1.530 0.020 1 260 38 47 ARG HD2 H 3.036 0.020 1 261 38 47 ARG HD3 H 3.036 0.020 1 262 38 47 ARG HE H 7.124 0.020 1 263 38 47 ARG N N 113.124 0.3 1 264 38 47 ARG NE N 84.099 0.3 1 265 39 48 GLU H H 8.613 0.020 1 266 39 48 GLU HA H 4.090 0.020 1 267 39 48 GLU HB2 H 1.940 0.020 1 268 39 48 GLU HB3 H 1.940 0.020 1 269 39 48 GLU HG2 H 2.310 0.020 2 270 39 48 GLU HG3 H 2.180 0.020 2 271 39 48 GLU N N 123.851 0.3 1 272 40 49 ALA H H 8.090 0.020 1 273 40 49 ALA HA H 4.970 0.020 1 274 40 49 ALA HB H 1.570 0.020 1 275 40 49 ALA N N 122.772 0.3 1 276 41 50 VAL H H 8.396 0.020 1 277 41 50 VAL HA H 4.449 0.020 1 278 41 50 VAL HB H 1.360 0.020 1 279 41 50 VAL HG1 H 0.600 0.020 2 280 41 50 VAL HG2 H 0.099 0.020 2 281 41 50 VAL N N 119.835 0.3 1 282 42 51 ILE H H 8.958 0.020 1 283 42 51 ILE HA H 4.915 0.020 1 284 42 51 ILE HB H 1.620 0.020 1 285 42 51 ILE HG12 H 1.410 0.020 2 286 42 51 ILE HG13 H 0.900 0.020 2 287 42 51 ILE HG2 H 0.740 0.020 1 288 42 51 ILE HD1 H 0.650 0.020 1 289 42 51 ILE N N 124.884 0.3 1 290 43 52 PHE H H 9.305 0.020 1 291 43 52 PHE HA H 5.290 0.020 1 292 43 52 PHE HB2 H 2.930 0.020 2 293 43 52 PHE HB3 H 3.130 0.020 2 294 43 52 PHE HD1 H 7.240 0.020 1 295 43 52 PHE HD2 H 7.240 0.020 1 296 43 52 PHE HE1 H 6.910 0.020 1 297 43 52 PHE HE2 H 6.910 0.020 1 298 43 52 PHE HZ H 7.260 0.020 1 299 43 52 PHE N N 126.985 0.3 1 300 44 53 LYS H H 8.944 0.020 1 301 44 53 LYS HA H 5.337 0.020 1 302 44 53 LYS HB2 H 1.980 0.020 2 303 44 53 LYS HB3 H 1.850 0.020 2 304 44 53 LYS HG2 H 1.400 0.020 1 305 44 53 LYS HG3 H 1.400 0.020 1 306 44 53 LYS HD2 H 1.640 0.020 1 307 44 53 LYS HD3 H 1.640 0.020 1 308 44 53 LYS HE2 H 2.950 0.020 1 309 44 53 LYS HE3 H 2.950 0.020 1 310 44 53 LYS N N 122.331 0.3 1 311 45 54 THR H H 9.020 0.020 1 312 45 54 THR HA H 5.177 0.020 1 313 45 54 THR HB H 4.727 0.020 1 314 45 54 THR HG2 H 1.198 0.020 1 315 45 54 THR N N 116.428 0.3 1 316 46 55 LYS H H 8.627 0.020 1 317 46 55 LYS HA H 4.210 0.020 1 318 46 55 LYS HB2 H 1.740 0.020 2 319 46 55 LYS HB3 H 2.030 0.020 2 320 46 55 LYS HG2 H 1.400 0.020 2 321 46 55 LYS HG3 H 1.500 0.020 2 322 46 55 LYS HD2 H 1.500 0.020 1 323 46 55 LYS HD3 H 1.500 0.020 1 324 46 55 LYS HE2 H 3.000 0.020 1 325 46 55 LYS HE3 H 3.000 0.020 1 326 46 55 LYS N N 119.542 0.3 1 327 47 56 LEU H H 7.715 0.020 1 328 47 56 LEU HA H 4.467 0.020 1 329 47 56 LEU HB2 H 1.783 0.020 2 330 47 56 LEU HB3 H 1.560 0.020 2 331 47 56 LEU HG H 1.590 0.020 1 332 47 56 LEU HD1 H 0.930 0.020 2 333 47 56 LEU HD2 H 0.870 0.020 2 334 47 56 LEU N N 117.667 0.3 1 335 48 57 ASP H H 8.026 0.020 1 336 48 57 ASP HA H 4.343 0.020 1 337 48 57 ASP HB2 H 3.007 0.020 2 338 48 57 ASP HB3 H 2.652 0.020 2 339 48 57 ASP N N 116.146 0.3 1 340 49 58 LYS H H 7.398 0.020 1 341 49 58 LYS HA H 4.561 0.020 1 342 49 58 LYS HB2 H 1.740 0.020 2 343 49 58 LYS HB3 H 1.820 0.020 2 344 49 58 LYS HG2 H 1.310 0.020 1 345 49 58 LYS HG3 H 1.310 0.020 1 346 49 58 LYS HD2 H 1.440 0.020 1 347 49 58 LYS HD3 H 1.440 0.020 1 348 49 58 LYS HE2 H 3.010 0.020 1 349 49 58 LYS HE3 H 3.010 0.020 1 350 49 58 LYS N N 117.192 0.3 1 351 50 59 GLU H H 8.453 0.020 1 352 50 59 GLU HA H 5.388 0.020 1 353 50 59 GLU HB2 H 1.960 0.020 2 354 50 59 GLU HB3 H 1.780 0.020 2 355 50 59 GLU HG2 H 2.000 0.020 2 356 50 59 GLU HG3 H 2.430 0.020 2 357 50 59 GLU N N 120.425 0.3 1 358 51 60 ILE H H 9.149 0.020 1 359 51 60 ILE HA H 4.495 0.020 1 360 51 60 ILE HB H 1.950 0.020 1 361 51 60 ILE HG12 H 1.230 0.020 2 362 51 60 ILE HG13 H 1.570 0.020 2 363 51 60 ILE HG2 H 1.120 0.020 1 364 51 60 ILE HD1 H 0.880 0.020 1 365 51 60 ILE N N 121.936 0.3 1 366 52 61 CYS H H 8.841 0.020 1 367 52 61 CYS HA H 5.060 0.020 1 368 52 61 CYS HB2 H 3.455 0.020 2 369 52 61 CYS HB3 H 2.798 0.020 2 370 52 61 CYS N N 125.890 0.3 1 371 53 62 ALA H H 9.833 0.020 1 372 53 62 ALA HA H 4.971 0.020 1 373 53 62 ALA HB H 1.320 0.020 1 374 53 62 ALA N N 127.119 0.3 1 375 54 63 ASP H H 8.396 0.020 1 376 54 63 ASP HA H 4.070 0.020 1 377 54 63 ASP HB2 H 2.600 0.020 2 378 54 63 ASP HB3 H 1.640 0.020 2 379 54 63 ASP N N 121.425 0.3 1 380 56 65 THR H H 8.236 0.020 1 381 56 65 THR HA H 4.040 0.020 1 382 56 65 THR HB H 4.200 0.020 1 383 56 65 THR HG2 H 1.180 0.020 1 384 56 65 THR N N 109.730 0.3 1 385 57 66 GLN H H 7.411 0.020 1 386 57 66 GLN HA H 4.210 0.020 1 387 57 66 GLN HB2 H 1.650 0.020 2 388 57 66 GLN HB3 H 1.850 0.020 2 389 57 66 GLN HG2 H 2.370 0.020 2 390 57 66 GLN HG3 H 2.230 0.020 2 391 57 66 GLN HE21 H 7.366 0.020 1 392 57 66 GLN HE22 H 6.969 0.020 1 393 57 66 GLN N N 120.704 0.3 1 394 57 66 GLN NE2 N 113.306 0.3 1 395 58 67 LYS H H 8.774 0.020 1 396 58 67 LYS HA H 3.763 0.020 1 397 58 67 LYS HB2 H 1.920 0.020 1 398 58 67 LYS HB3 H 1.920 0.020 1 399 58 67 LYS HG2 H 1.473 0.020 1 400 58 67 LYS HG3 H 1.473 0.020 1 401 58 67 LYS HD2 H 1.750 0.020 2 402 58 67 LYS HD3 H 1.650 0.020 2 403 58 67 LYS HE2 H 3.060 0.020 1 404 58 67 LYS HE3 H 3.060 0.020 1 405 58 67 LYS N N 126.044 0.3 1 406 59 68 TRP H H 8.279 0.020 1 407 59 68 TRP HA H 4.282 0.020 1 408 59 68 TRP HB2 H 3.050 0.020 2 409 59 68 TRP HB3 H 3.010 0.020 2 410 59 68 TRP HD1 H 7.580 0.020 1 411 59 68 TRP HE1 H 10.009 0.020 1 412 59 68 TRP HE3 H 6.350 0.020 1 413 59 68 TRP HZ2 H 7.380 0.020 1 414 59 68 TRP HZ3 H 6.510 0.020 1 415 59 68 TRP HH2 H 6.940 0.020 1 416 59 68 TRP N N 115.568 0.3 1 417 59 68 TRP NE1 N 129.477 0.3 1 418 60 69 VAL H H 5.700 0.020 1 419 60 69 VAL HA H 2.790 0.020 1 420 60 69 VAL HB H 1.780 0.020 1 421 60 69 VAL HG1 H -0.670 0.020 2 422 60 69 VAL HG2 H 0.420 0.020 2 423 60 69 VAL N N 122.600 0.3 1 424 61 70 GLN H H 7.199 0.020 1 425 61 70 GLN HA H 3.960 0.020 1 426 61 70 GLN HB2 H 2.140 0.020 2 427 61 70 GLN HB3 H 2.080 0.020 2 428 61 70 GLN HG2 H 2.400 0.020 2 429 61 70 GLN HG3 H 2.280 0.020 2 430 61 70 GLN HE21 H 6.604 0.020 1 431 61 70 GLN HE22 H 7.369 0.020 1 432 61 70 GLN N N 118.577 0.3 1 433 61 70 GLN NE2 N 110.462 0.3 1 434 62 71 ASP H H 8.642 0.020 1 435 62 71 ASP HA H 4.429 0.020 1 436 62 71 ASP HB2 H 2.880 0.020 2 437 62 71 ASP HB3 H 2.780 0.020 2 438 62 71 ASP N N 120.010 0.3 1 439 63 72 PHE H H 8.551 0.020 1 440 63 72 PHE HA H 4.550 0.020 1 441 63 72 PHE HB2 H 3.530 0.020 2 442 63 72 PHE HB3 H 2.900 0.020 2 443 63 72 PHE HD1 H 7.010 0.020 1 444 63 72 PHE HD2 H 7.010 0.020 1 445 63 72 PHE HE1 H 7.440 0.020 1 446 63 72 PHE HE2 H 7.440 0.020 1 447 63 72 PHE HZ H 7.380 0.020 1 448 63 72 PHE N N 122.392 0.3 1 449 64 73 MET H H 8.392 0.020 1 450 64 73 MET HA H 3.680 0.020 1 451 64 73 MET HB2 H 2.390 0.020 2 452 64 73 MET HB3 H 2.090 0.020 2 453 64 73 MET HG2 H 2.280 0.020 2 454 64 73 MET HG3 H 0.610 0.020 2 455 64 73 MET HE H 1.790 0.020 1 456 64 73 MET N N 119.547 0.3 1 457 65 74 LYS H H 7.627 0.020 1 458 65 74 LYS HA H 4.129 0.020 1 459 65 74 LYS HB2 H 1.980 0.020 2 460 65 74 LYS HB3 H 1.770 0.020 2 461 65 74 LYS HG2 H 1.520 0.020 2 462 65 74 LYS HG3 H 1.660 0.020 2 463 65 74 LYS HD2 H 1.700 0.020 1 464 65 74 LYS HD3 H 1.700 0.020 1 465 65 74 LYS HE2 H 3.020 0.020 1 466 65 74 LYS HE3 H 3.020 0.020 1 467 65 74 LYS N N 116.788 0.3 1 468 66 75 HIS H H 7.874 0.020 1 469 66 75 HIS HA H 4.420 0.020 1 470 66 75 HIS HB2 H 3.444 0.020 2 471 66 75 HIS HB3 H 3.256 0.020 2 472 66 75 HIS HD2 H 6.700 0.020 1 473 66 75 HIS HE1 H 7.220 0.020 1 474 66 75 HIS N N 117.397 0.3 1 475 67 76 LEU H H 8.131 0.020 1 476 67 76 LEU HA H 4.097 0.020 1 477 67 76 LEU HB2 H 2.220 0.020 2 478 67 76 LEU HB3 H 1.760 0.020 2 479 67 76 LEU HG H 2.010 0.020 1 480 67 76 LEU HD1 H 1.110 0.020 2 481 67 76 LEU HD2 H 0.850 0.020 2 482 67 76 LEU N N 119.706 0.3 1 483 68 77 ASP H H 8.598 0.020 1 484 68 77 ASP HA H 4.589 0.020 1 485 68 77 ASP HB2 H 2.880 0.020 2 486 68 77 ASP HB3 H 2.760 0.020 2 487 68 77 ASP N N 121.152 0.3 1 488 69 78 LYS H H 7.574 0.020 1 489 69 78 LYS HA H 4.210 0.020 1 490 69 78 LYS HB2 H 1.860 0.020 2 491 69 78 LYS HB3 H 1.910 0.020 2 492 69 78 LYS HG2 H 1.480 0.020 1 493 69 78 LYS HG3 H 1.480 0.020 1 494 69 78 LYS HD2 H 1.580 0.020 1 495 69 78 LYS HD3 H 1.580 0.020 1 496 69 78 LYS HE2 H 3.020 0.020 1 497 69 78 LYS HE3 H 3.020 0.020 1 498 69 78 LYS N N 118.966 0.3 1 499 70 79 LYS H H 7.873 0.020 1 500 70 79 LYS HA H 4.290 0.020 1 501 70 79 LYS HB2 H 1.890 0.020 2 502 70 79 LYS HB3 H 1.820 0.020 2 503 70 79 LYS HG2 H 1.480 0.020 1 504 70 79 LYS HG3 H 1.480 0.020 1 505 70 79 LYS HD2 H 1.700 0.020 1 506 70 79 LYS HD3 H 1.700 0.020 1 507 70 79 LYS HE2 H 3.005 0.020 1 508 70 79 LYS HE3 H 3.005 0.020 1 509 70 79 LYS N N 120.248 0.3 1 510 71 80 THR H H 7.959 0.020 1 511 71 80 THR HA H 4.350 0.020 1 512 71 80 THR HB H 4.280 0.020 1 513 71 80 THR HG2 H 1.260 0.020 1 514 71 80 THR N N 113.405 0.3 1 515 72 81 GLN H H 8.201 0.020 1 516 72 81 GLN HA H 4.421 0.020 1 517 72 81 GLN HB2 H 2.030 0.020 2 518 72 81 GLN HB3 H 2.140 0.020 2 519 72 81 GLN HG2 H 2.395 0.020 1 520 72 81 GLN HG3 H 2.395 0.020 1 521 72 81 GLN HE21 H 7.561 0.020 1 522 72 81 GLN HE22 H 6.892 0.020 1 523 72 81 GLN N N 122.343 0.3 1 524 72 81 GLN NE2 N 111.712 0.3 1 525 73 82 THR H H 8.192 0.020 1 526 73 82 THR HA H 4.607 0.020 1 527 73 82 THR HB H 4.170 0.020 1 528 73 82 THR HG2 H 1.270 0.020 1 529 73 82 THR N N 118.201 0.3 1 530 75 84 LYS H H 8.332 0.020 1 531 75 84 LYS HA H 4.320 0.020 1 532 75 84 LYS HB2 H 1.850 0.020 2 533 75 84 LYS HB3 H 1.750 0.020 2 534 75 84 LYS HG2 H 1.460 0.020 1 535 75 84 LYS HG3 H 1.460 0.020 1 536 75 84 LYS HD2 H 1.700 0.020 1 537 75 84 LYS HD3 H 1.700 0.020 1 538 75 84 LYS HE2 H 3.020 0.020 1 539 75 84 LYS HE3 H 3.020 0.020 1 540 75 84 LYS N N 122.409 0.3 1 541 76 85 LEU H H 7.870 0.020 1 542 76 85 LEU HA H 4.210 0.020 1 543 76 85 LEU HB2 H 1.590 0.020 1 544 76 85 LEU HB3 H 1.590 0.020 1 545 76 85 LEU HG H 1.590 0.020 1 546 76 85 LEU HD1 H 0.860 0.020 2 547 76 85 LEU HD2 H 0.900 0.020 2 548 76 85 LEU N N 130.000 0.3 1 stop_ save_